Agents for gene therapy of cerebrovascular disorders

ABSTRACT

The present invention provides novel methods for treating cerebrovascular disorders, in which HGF is overexpressed by introducing an HGF gene. The methods of this invention using an HGF gene enable active treatment of cerebrovascular disorders, such as cerebral infarction, by gene transfer, and enable the maintenance of neuronal function and the suppression of infarcted areas in patients for whom appropriate treatment methods were unavailable until now.

TECHNICAL FIELD

The present invention relates to the treatment or prevention ofcerebrovascular disorders by using a hepatocyte growth factor (HGF)gene. More specifically, the present invention relates to agents fortreatment or prevention, that comprise an HGF gene as an activeingredient, and methods that comprise the step of administering such anagent to a target site.

BACKGROUND ART

Hepatocyte growth factor (HGF) is a cytokine discovered as a factor thatcauses proliferation of hepatic parenchymal cells in vitro (Biochem.Biophys. Res. Commun. 122: 1450 (1984); Proc. Natl. Acad. Sci. USA 83:6489 (1986); FEBS Letters 22: 231 (1987); Nature 342: 440-443 (1989);Proc. Natl. Acad. Sci. USA 87: 3200 (1991)). HGF is aplasminogen-related and mesenchyme-derived pleiotropic growth factor,and is known to regulate cell growth and cell motility in various typesof cells (Nature 342: 440-443 (1989); Biochem. Biophys. Res. Commun.239: 639-644 (1997); J. Biochem. Tokyo 119: 591-600 (1996)). HGF is alsoknown to be an important factor regulating embryogenesis and morphogenicprocesses in the regeneration of a number of organs (Exp. Cell Res. 196:114-120 (1991); Proc. Natl. Acad. Sci. USA 90: 1937-1941 (1993); GeneTherapy 7: 417-427 (2000)). HGF not only contributes in vivo as a liverregeneration factor in the repair and regeneration of damaged liver, ithas also been shown to possess an angiogenic effect, and can play animportant role in the treatment or prevention of ischemic or arterialdiseases (Symp. Soc. Exp. Biol. 47 cell behavior 227-234 (1993); Proc.Natl. Acad. Sci. USA 90: 1937-1941 (1993); Circulation 97: 381-390(1998)).

Since HGF displays a variety of functions, including angiogenicfunctions, as described above, various studies for its use inpharmaceutical agents have been carried out (Jikken Igaku (ExperimentalMedicine) (supplementary volume) 10(3): 330-339 (1992)). For example,there are reports of using HGF as an:

-   -   anticancer agent (Unexamined Published Japanese Patent        Application No. (JP-A) Hei 6-25010);    -   therapeutic agent for lung injuries (JP-A Hei 6-172207);    -   agent for relieving the side effects of cancer therapy (JP-A Hei        6-340546);    -   agent for enhancing the growth of epithelial cells (JP-A Hei        7-179356);    -   agent for relieving the side effects of immunosuppressants (JP-A        Hei 7-258111);    -   therapeutic agent for fulminant hepatitis (JP-A Hei 10-167982);    -   therapeutic agent for myocardial infarctions (JP-A Hei        11-246433);    -   therapeutic agent for arterial diseases (JP-A Hei 8-295634);    -   therapeutic agent for obesity (JP-A Hei 10-279500);    -   therapeutic agent for dilated cardiomyopathy (JP-A Hei 11-1439);    -   therapeutic agent for amyotrophic lateral sclerosis (JP-A        2002-87983);    -   preventive agent for pulmonary fibrosis (JP-A Hei 8-268906);    -   therapeutic agent for cartilage disorders (JP-A Hei 8-59502);    -   collagen degradation promoting agent (JP-A Hei 7-300426);    -   therapeutic agent for gastroduodenal diseases (JP-A Hei        7-138183);    -   therapeutic agent for cranial nerve disorders (JP-A Hei        7-89869);    -   therapeutic agent for acute renal failure (Published Japanese        Translation of International Publication No. 2001-516358);    -   therapeutic agent for ischemic diseases/arterial diseases (WO        00/07615);    -   therapeutic agent for diabetes (WO 98/32458);    -   external agent for hair (JP-A Hei 5-213721);    -   dermal cosmetics (JP-A Hei 5-213733);    -   agent for promoting hair growth (JP-A Hei 5-279230);    -   agent for increasing megakaryocytes (JP-A Hei 7-101876);    -   differentiation-inducing agent (JP-A 2002-78482);    -   therapeutic agent for renal glomerular diseases (JP-A Hei        9-87199);    -   therapeutic agent for cachexia (JP-A Hei 10-316584);    -   therapeutic agent for multiple organ failure (JP-A Hei        10-310535);    -   therapeutic agent for ischemic diseases (WO 96/32960);    -   agent for cell proliferation and differentiation (Published        Japanese Translation of International Publication No. Hei        10-503923);    -   agent for growth and differentiation of hematopoietic cells        (Published Japanese Translation of International Publication No.        Hei 10-509951);    -   agent for improving neuropathy (JP-A Hei 7-41429); and    -   therapeutic agent for hypoglycemia and glycogen diseases (JP-A        Hei 10-7586).

Typically, proteinaceous formulations are administered intravenously. Inischemic disease models, HGF administration is exemplified byintravenous and intra-arterial administration (Circulation 97: 381-390(1998)). Although intravenous or intra-arterial HGF administration hasbeen shown to be effective in this type of animal model, no conclusionshave been reached regarding effective methods of administration,dosages, and such of HGF for specific diseases. In particular, HGF'sshort half-life in the blood has emerged as a problem when applying HGFproteins as pharmaceutical agents. Since the half-life of HGF in theblood is short as ten minutes, it has been difficult to maintain HGFconcentration in the blood at a level where HGF functions sufficiently.Furthermore, another challenge has been raised regarding the delivery ofan effective amount of HGF to affected sites.

Thanks to remarkable technological progress in the field of molecularbiology, gene therapy involving the introduction of genes into cells isnow possible. Gene therapy can generally be used in various medicaltreatments (Science 256: 808-813 (1992); Anal. Biochem. 162: 156-159(1987)). Selecting an appropriate vector for gene transfer isparticularly important for successful gene therapy. So far, vectorsderived from viruses such as adenoviruses have been suggested for use ingene transfer.

However, viral vectors have also been suggested to potentially have thefollowing dangers:

-   -   viral infection-associated toxicity;    -   development of viral pathogenicity associated with the depressed        immunological function of the host;    -   mutagenic or carcinogenic nature of the viruses; and such.

As an alternative to methods using viral vectors, in vivo gene transfermethods using liposomes together with viral outer membranes, orHVJ-liposome-mediated gene transfer methods, have been developed(Science 243: 375-378 (1989); Anal. NY Acad. Sci. 772: 126-139 (1995)).In vivo gene transfers into various tissues, including the liver,kidney, vascular wall, heart, and brain, have been successfullyaccomplished using these methods (Gene Therapy 7: 417-427 (2000);Science 243: 375-378 (1989); Biochem. Biophys. Res. Commun. 186: 129-134(1992); Proc. Natl. Acad. Sci. USA 90: 8474-8478 (1993); Am. J. Physiol.271 (Regulatory Integrative Comp. Physiol. 40):R1212-R1220 (1996)).

However, since methods using HVJ-liposomes require different vehicles,such as viruses and liposomes, they are complicated to prepare.Furthermore, fusing HVJ virions with liposomes makes the averagediameter of the resultant particles 1.3 times that of a viral particle.This increased diameter is known to reduce cell fusion activity to 10%or less of that of a wildtype virus, and there are tissues to which genetransfer is impossible or inefficient. Accordingly, a gene transfermethod that uses viral envelope vectors was developed as a method thatallows safer and more efficient gene therapy (Japanese PatentApplication No. 2001-026185/JP-A 2001-286282). In this method, aninactivated virus, which cannot replicate viral proteins, is used as aviral envelope, and genes are enclosed within this viral envelope. Thevector can be used for gene transfer into cultured cells, biologicaltissues, and such. The use of such viral envelope vectors is known toenable safe and highly efficient gene transfer into the liver, skeletalmuscle, uterus, brain, eyes, carotid arteries, skin, blood vessels,lungs, heart, kidneys, spleen, cancer tissues, nerves, B lymphocytes,respiratory organ tissue, suspended cells, and such.

Cerebrovascular disorders, represented by cerebral infarctions andintracerebral hemorrhages, are important diseases that are also sociallysignificant. Although mortality due to cerebrovascular disorders hasdeclined in recent years, such diseases are still highly ranked causesof death. Patients affected with the aftereffects of an ischemiccerebrovascular disorder, and hospitalized or outpatients being treatedby clinical institutions, still continue to increase.

Generally, a cerebral infarction is a condition whereby the brain tissuebecomes irreversibly necrotic, due to ischemic lesions caused byocclusion, or by a decrease of perfusion pressure in the cerebral arteryor carotid artery. Cerebral infarctions can be categorized intofollowing three major groups (Manabe, H., and Omae, T. Ed. “Nou-KekkanShogai (Cerebrovascular disorder)” Life Science Publishing, p54-55(1992); Imura, H. et al. Ed., “Saishin Naikagaku Taikei Dai 66 KanNou-Kekkan Shogai (Integrated handbook of internal medicine, Vol. 66,Cerebrovascular disorder)” Nakayama Shoten, p28 (1996)):

-   -   (1) cerebral thrombosis, in which ischemic necrosis occurs in        the cerebral tissue as a result of arterial occlusion, where the        arterial occlusion is caused by increased blood viscosity,        decreased perfusion pressure, or such, arising from sclerotic        lesions in the cerebral artery;    -   (2) cerebral embolism, in which an embolus forms in the cerebral        artery due to an intracardiac thrombus or, although rare, due to        a thrombus detached from the arterial wall; and    -   (3) hemodynamic infarction, caused by reduced blood flow to the        peripheral brain tissue due to the constriction or occlusion of        the cephalic artery or intracranial cerebral artery.

In cerebral infarction, a cerebral edema occurs within a few hours ofthe onset of disease, and this condition continues for approximately oneweek after onset. Thereafter, the edema gradually decreases, but becomesfixed as an infarcted area within one to three months after onset. Thecerebral edema causes the volume of the brain to increase. Since thebrain is covered with a hard cranium, when the volume of the brainexceeds a certain limit due to the cerebral edema, rapid increase intissue pressure and intra-cranial pressure results. Thus, brain damageworsens, and thereafter, the extent of the infarcted area is fixed(Inamura, K., Terashi, A. “Nippon Rinsho, Dai 51 Kan, CT, MRI Jidai noNo-Sotchu Gaku, Jo-Kan (Nippon Rinsho, Vol. 51, Cerebral Apoplexy in theAge of CT and MRI, No. 1)” Nippon Rinsho, p231-239 (1993)). When aninfarction occurs in a section of the brain, those functions carried bythe affected part, such as cognition, perception, sense, and memory, arelost.

To date, nerve cells have been clinically recognized as being vulnerableto ischemia. Certain types of nerve cells are damaged when placed underischemic conditions for only a few minutes, and these cells subsequentlydie. In hippocampal formations and pyramidal cells under ischemicconditions, a conduction block occurs after significant nerve excitationassociated with depolarization. Subsequently, cell functions are lostdue to the cytotoxicity caused by increased levels of extracellularglutamic acid, intracellular calcium ion, free radicals, and such, andthe cells eventually die. If the irreversible changes caused by ischemiacan be appropriately treated in an acute stage, it is thought thatmortality rates can be improved, and aftereffects alleviated. Currenttreatment for cerebral infarction involves administering antiplateletagents, agents for improving cerebral circulation and metabolism, andsuch. Among these antiplatelet agents, pharmaceutical agents effectivefor treating acute stage cerebral thrombosis exist. However, since theseagents promote hemorrhagic cerebral infarction in patients sufferingfrom cerebral hemorrhages or cerebral infarctions that develop symptomssimilar to cerebral thrombosis, their use in these patients iscontraindicated, and the disease type must be carefully diagnosed whenusing such agents.

During the acute stage, the use of agents for improving cerebralcirculation used for administration during the chronic stage, which isapproximately one month after the cerebral infarction attack, isconsidered unfavorable (Kameyama, M. Ed. “Nou-sotchu Chiryou Manyuaru(Treatment Manual of Cerebral Apoplexy)” Igaku-Shoin, p172-173 (1991)).Reperfusion therapy such as thrombolytic therapy, bypass surgery,thrombendarterectomy, and embolectomy are used as alternative methods oftreatment during the hyperacute stage, in order to re-establish bloodflow to regions where cells have not yet died. However, when the braintissue is already irreversibly damaged by the cerebral infarction, thesubsequent re-establishment of blood flow is problematic due to thedanger of aggravating tissue injury, such as increasing the hemorrhagicinfarction and cerebral edema (Okada, Y., “Shinkei Kenkyu no Shinpo(Progress in Neurologic Research)” Vol. 40, No. 4, p. 655-665,Igaku-Shoin, (1996); Takahasi, A. “medicina” Vol. 32, No. 11, p.2261-2263, Igaku-Shoin, (1991)).

At present, the pharmaceutical agents used in the acute stages ofcerebral infarctions are risky in that they may cause hemorrhagicinfarctions and ischemic/reperfusion injuries. Furthermore, these agentsare not completely satisfactory due to other problems such as thelimited pathogenic conditions targeted, and the limited period overwhich administration is expected to be therapeutically effective.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to provide pharmaceuticalagents that can reduce the degree of brain injury caused by cerebralischemic/reperfusion injuries associated with reperfusion after bloodflow to the brain has been cut off. The HGF gene differs from otherangiogenesis factors, such as VEGF, in that it does not increase thepermeability of newly produced blood vessels. In cerebrovasculardisorders in particular, increased vascular permeability increases thedanger of injury to cerebral tissues by cerebral edema and increasedintracerebral pressure. Thus, therapeutic and preventive methods usingHGF that do not increase vascular permeability, are advantageouscompared to methods using other angiogenesis factors.

An objective of the present invention is to provide therapeutic orpreventive agents that use an HGF gene, against cerebrovasculardisorders, and to provide methods of treating and preventingcerebrovascular disorders using these pharmaceutical agents. Morespecifically, the present invention is summarized as follows.

-   (1) An agent for treating or preventing a cerebrovascular disorder,    wherein the agent comprises an HGF gene.-   (2) The agent of (1), wherein the cerebrovascular disorder is a    cerebral infarction.-   (3) The agent of (1) or (2), wherein the agent is in the form of a    tablet, pill, sugar-coated tablet, capsule, liquid, gel, ointment,    syrup, slurry, or suspension.-   (4) The agent of any one of (1) to (3), wherein the agent is used to    transfer a gene into a cell by a method employing a viral envelope    vector, internal type liposome, electrostatic type liposome,    HVJ-liposome, or improved HVJ-liposome, a receptor-mediated gene    transfer method, a method for transferring a nucleic acid molecule    along with a carrier into a cell by using a particle gun, a direct    introduction method using a naked-DNA, or an introduction method    using a cationic polymer.-   (5) The agent of any one of (1) to (3), wherein the agent is used to    transfer a gene into a cell by employing an HVJ-envelope.-   (6) A method for treating or preventing a cerebrovascular disorder,    wherein the method comprises the step of introducing an HGF gene    into a mammal.-   (7) The method of (6), wherein the cerebrovascular disorder is a    cerebral infarction.-   (8) The method of (6) or (7), wherein the method comprises the step    of introducing an HGF gene into a mammal two to three times by    employing an HVJ-envelope.-   (9) Use of an HGF gene to produce an agent for treating or    preventing a cerebrovascular disorder.-   (10) The use of the HGF gene of (9), wherein the cerebrovascular    disorder is a cerebral infarction.

The term “HGF gene” as used in this invention refers to a nucleic acidmolecule that can express an HGF (an HGF protein). Herein, the term“nucleic acid molecule” refers to molecules such as DNAs, RNAs, cDNAs,and mRNAs. Specifically, cDNAs that encode HGF are described in, forexample, Nature 342: 440 (1989), U.S. Pat. No. 2,777,678, and Biochem.Biophys. Res. Commun. 163: 967 (1989). The nucleotide sequences of cDNAsencoding HGF are described in the aforementioned literature, and arealso registered in databases such as GenBank. Based on this sequenceinformation, cDNAs of HGF can be cloned by using appropriate sequencesegments as PCR primers, and by performing RT-PCR using, for example,mRNAs derived from the liver or leukocytes. One skilled in the art canreadily perform such cloning by following fundamental texts, such asMolecular Cloning 2nd edition (Cold Spring Harbor Laboratory Press)(1989). Furthermore, by screening genomic DNA libraries, genomic DNAscan be isolated.

The HGF genes of this invention are not limited to these cDNAs andgenomic DNAs. As long as a gene encodes a protein that, when expressed,has practically the same function as HGF, the gene can be used as an HGFgene of this invention. More specifically, the HGF genes that can beused in this invention encompass 1) nucleic acids that hybridize understringent conditions with an aforementioned cDNA, or 2) nucleic acidsencoding proteins that comprise an amino acid sequence of a proteinencoded by an aforementioned cDNA, in which one or more (preferablyseveral) amino acids are deleted, substituted, added, and/or inserted,so long as the proteins encoded by such nucleic acids have a function ofHGF. The nucleic acids of the aforementioned 1) and 2) can be readilyobtained by methods such as site-directed mutagenesis, PCR methods (see,Current Protocols in Molecular Biology edit. Ausubel et al. (1987) JohnWiley & Sons, Sections 6.1-6.4), or conventional hybridization methods(see, Current Protocols in Molecular Biology edit. Ausubel et al. (1987)John Wiley & Sons, Sections 6.3-6.4).

More specifically, by using a known sequence of an HGF gene, or aportion thereof as a probe; or by using an oligonucleotide thatspecifically hybridizes with that DNA sequence as a primer, thoseskilled in the art can isolate nucleic acids that hybridize with thatDNA sequence. Stringent hybridization conditions for obtaining nucleicacids that encode proteins functionally equivalent to known HGF arenormally “1×SSC at 37° C.” and such, more stringently “0.5×SSC and 0.1%SDS at 42° C.” and such, and even more stringently “0.1×SSC and 0.1% SDSat 65° C.” and such. When more stringent hybridization conditions areused, nucleic acids comprising sequences more homologous to the probesequence can be isolated. However, the SSC, SDS, and temperatureconditions recited herein are only examples. One skilled in the art canreadily set conditions that constitute the same degree of stringency asthe above-described, by considering the above-mentioned conditions andother conditions that determine the stringency of hybridization, such asprobe concentration, probe length, and reaction time.

The amino acid sequences of the proteins encoded by the nucleic acidsisolated by the above-mentioned hybridization methods or PCR methodsusually show high homology to conventionally known HGF proteins. Theterm “high homology” refers to a sequence homology of at least 50% ormore, more preferably 70% or more, even more preferably 90% or more (forexample, 95% or more). The sequence identity of amino acid sequences andnucleotide sequences can be determined using Karlin and Altschul's BLASTalgorithm (Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993)). Programssuch as BLASTN and BLASTX have been developed based on this algorithm(Altschul et al. J. Mol. Biol. 215: 403-410 (1990)). When nucleotidesequences are analyzed using BLASTN, based on BLAST, parameters are set,for example, at score=100 and wordlength=12. When amino acid sequencesare analyzed by using BLASTX, based on BLAST, parameters are set, forexample, at score=50 and wordlength=3. When using the BLAST and GappedBLAST programs, the default parameters for the respective programs areused. Specific techniques for these analytical methods are well known(http://www.ncbi.nlm.nih.gov).

As described above, the HGF genes used in this invention can benaturally occurring or artificial nucleic acids, as long as the proteinsencoded by the genes comprise HGF activity. HGF has the activity ofpromoting the in vitro proliferation of hepatic parenchymal cells.Therefore, it is possible to determine whether the proteins encoded bythe nucleic acids obtained by the above-described hybridization methodsor such, or the proteins encoded by the nucleic acids of a modifiednaturally occurring HGF gene, have HGF activity, by, for example,investigating the effect of these proteins on the in vitro proliferationof hepatic parenchymal cells.

The gene transfer methods, transfer forms, transfer amounts, and suchused in the gene therapies of this invention are described below.

Methods for administering gene therapy agents comprising an HGF gene asan active ingredient can be classified into two groups: methods usingnon-viral vectors; and methods using viral vectors. The preparationmethods, administration methods, and such for these vectors aredescribed in detail in experiment manuals (Jikken Igaku (ExperimentalMedicine) Supplementary Volume, “Idenshichiryo no Kisogijyutsu(Fundamental Techniques for Gene Therapy)”, Yodosha, 1996; Jikken Igaku(Experimental Medicine) Supplementary Volume, “Idenshidonyu &Hatsugenkaiseki Jikkenho (Experimental Methods for Gene Transfer &Expression Analysis)”, Yodosha, 1997; “Idenshi-chiryo Kaihatsu KenkyuHandbook (Handbook of Gene Therapy Research and Development)”, NihonIdenshichiryo Gakkai (The Japan Society of Gene Therapy) Edition, NTS,1999). Such vectors and methods are specifically described below.

A recombinant vector, where a target gene has been inserted into aconventional non-viral gene expression vector, can be used to introducea target gene into cells and tissues by the methods shown below.

Examples of methods for transferring genes into cells are: lipofectionmethods, calcium-phosphate co-precipitation methods, DEAE-dextranmethods, methods of direct infusion of DNA using a glass capillary tube,etc. Methods for transferring genes into tissues include methods usingvirus envelope vectors, internal type liposomes, electrostatic typeliposomes, HVJ-liposomes, improved type HVJ-liposomes (HVJ-AVEliposomes), receptor-mediated gene transfer methods, methods fortransferring carriers (such as metal particles) along with DNAs usingparticle guns, methods for directly introducing naked-DNAs, introductionmethods using positively charged polymers, etc.

The aforementioned HVJ-liposomes are constructed by incorporating a DNAinto a liposome formed by a lipid bilayer, then fusing this liposomewith an inactivated Sendai virus (hemagglutinating virus of Japan; HVJ).The use of HVJ-liposomes is characterized by extremely high cellmembrane fusion compared to conventional liposome methods, and is one ofthe especially preferred forms of introduction. Methods for preparingHVJ-liposomes are described in detail in, for example, ExperimentalMedicine Supplementary Volume, “Idenshichiryo no Kisogijyutsu(Fundamental Techniques of Gene Therapy)”, Yodosha, 1996; ExperimentalMedicine Supplementary Volume, “Idenshidonyu & Hatsugenkaiseki Jikkenho(Experimental Methods for Gene Transfer & Expression Analysis)”, Yodosha(1997); J. Clin. Invest. 93: 1458-1464 (1994); Am. J. Physiol. 271:R1212-1220(1996). Methods for using the HVJ-liposome are described in,for example, Molecular Medicine 30: 1440-1448 (1993); Jikken Igaku(Experimental Medicine), 12: 1822-1826 (1994); and Tanpakushitsu KakusanKouso (Protein, Nucleic Acid, and Enzyme), 42, 1806-1813 (1997); andpreferably described in Circulation 92 (Suppl. II): 479-482 (1995).

Furthermore, methods using viral envelopes are particularly preferredmethods for administering an HGF gene of this invention. Viral envelopescan be prepared by mixing a purified virus with an expression vector ofinterest in the presence of a surfactant, or by freezing and thawing amixture of a virus and an expression vector (Japanese Patent ApplicationNo. 2001-026185/JP-A 2001-286282).

The viruses that can be used in the methods using viral envelopes areviruses belonging to families such as the retrovirus, togavirus,coronavirus, flavivirus, paramyxovirus, orthomyxovirus, bunyavirus,rhabdovirus, poxvirus, herpesvirus, baculovirus, and hepadnavirusfamilies, and are especially preferably HVJs. Herein, these viruses canbe either wild-type or recombinant viruses. In particular, a recombinantHVJ reported by Hasan, M. K. et al. (J. General Virol. 78: 2813-2820(1997)), Yonemitsu, Y. et al. (Nature Biotech. 18: 970-973 (2000)), orsuch may be used as an HVJ.

While the Z strain (available from ATCC) of HVJ is generally preferablefor use in the methods using HVJ-liposomes or HVJ-envelopes,fundamentally, other HVJ strains (for example, ATCC VR-907 and ATCCVR-105) can also be used. In the methods for preparing a viral envelope,purified viruses can be inactivated by UV irradiation and such, and thenmixed with a desired expression vector. Surfactants that may be used formixing the virus and expression vector are, for example, octylglucoside,Triton X-100, CHAPS, and NP-40. Viral envelope vectors prepared in thismanner can be introduced by injection or such into tissues to betargeted for therapy or disease prevention. Furthermore, by freezing at−20° C., the viral envelope vectors can be stored for at least two tothree months.

The expression vectors that may be used herein can be any expressionvectors, so long as they can express a desired gene in vivo. Examples ofthe expression vectors are PCAGGS (Gene 108: 193-200 (1991)), pBK-CMV,pcDNA3.1, and pZeoSV (Invitrogen, Stratagene)

Representative methods for gene transfer with viral vectors are thosemethods using viral vectors such as recombinant adenoviruses andretroviruses. More specifically, a subject gene can be transferred intocells by the steps of: introducing the gene into DNA or RNA viruses suchas detoxicated retroviruses, adenoviruses, adeno-associated viruses,herpesviruses, lentiviruses, vaccinia viruses, poxviruses, polioviruses,sindbis viruses, Sendai viruses, SV40, or human immunodeficiency viruses(HIV) (see Pharmacol. Ther. 80: 35-47 (1998); Front. Biosci. 4: E26-33(1999); J. Recep. Signal. Transduct. Res. 19: 673-686); and theninfecting cells with the resultant recombinant virus.

The infection efficiency of adenovirus vectors is much greater than theother aforementioned viral vectors. Thus, from this viewpoint, the useof an adenovirus vector system is preferred.

Methods for introducing an agent of the present invention during genetherapy include: the in vivo introduction of a gene therapy agentdirectly into the body; and the ex vivo introduction of a gene therapyagent into a cell harvested from the body, followed by reintroduction ofthe modified cell into the body (Nikkei Science, April 1994, 20-45;Gekkann Yakuji 36 (1), 23-48, 1994; Jikken Igaku (Experimental Medicine)Supplementary Volume, 12 (15), 1994; “Idenshi-chiryo Kaihatsu KenkyuHandbook (Handbook of Gene Therapy Research and Development)”, NihonIdenshichiryo Gakkai eds. (The Japan Society of Gene Therapy) Edition,NTS, 1999). In vivo methods are particularly preferred in the presentinvention.

Various formulations, (for example, liquid preparations), suited to eachof the above-mentioned administration methods may be adopted as the formof the preparations. For example, an injection comprising a gene as anactive ingredient can be prepared by conventional methods, which mightinclude dissolving a gene in an appropriate solvent (e.g. a buffersolution, such as PBS, physiological saline, and sterilized water),sterilizing by filtration as necessary, and then loading into a sterilecontainer. Conventional carriers or such may be added to injectionagents as required. Alternatively, liposome preparations, such aspreparations comprising HVJ-liposome, can be prepared as suspensions,frozen agents, or centrifugally concentrated frozen agents.

For the therapeutic or preventive agents of this invention, an HGF genecan be used as the single active ingredient, and can also be usedtogether with other known factors having angiogenic functions. Forexample, factors such as VEGF and EGF have been reported to possessangiogenic functions, thus genes encoding these factors may beconcurrently applied. Furthermore, since growth factors such as EGF havebeen reported to repair cell lesions in various tissues, genes encodinga variety of growth factors may be concurrently used as necessary. Fromthe description of the present invention, it is clear to those skilledin the art that the following agents can be used, as required, incombination with the HGF gene in the therapeutic or preventive agents ofthe present invention: other pharmaceutical agents having therapeutic orpreventive effects against a cerebrovascular disorder to be treated orprevented; and substances that stabilize or enhance HGF (for example,heparin-like substances (JP-A Hei 10-158190) and oligosaccharides (JP-AHei 5-301824)). If these additional pharmaceutical agents and substancesare encoded by genes, the genes encoding them may be administeredtogether with the HGF gene in the therapeutic or preventive agents ofthis invention.

Appropriate administration methods and sites to be administered can beselected for the therapeutic and preventive agents of the presentinvention depending on the disorders, symptoms, or such to be treated.Parenteral administration is particularly preferred. The cisterna magnaand lumbar spine are especially preferable administration sites. Thecisterna magna or lumbar spine is punctured into the meningeal, and thenan appropriate amount of spinal fluid is collected to confirm thepuncture site, and to prevent an increase in intracranial pressure. Thetherapeutic or preventive agent is then administered. For example, amethod for administering HVJ-liposome complex using a cannula, reportedby Hayashi, K. et al. (Gene Therapy 8: 1167-73 (2001)), could be appliedto administer the therapeutic or preventive agents of this inventioninto the cisterna magna.

The effect of the therapeutic or preventive agents of this invention isconsidered to be caused by using HGF gene administration to sustainneurons by suppressing apoptosis in the so-called “penumbra” regionaround the focus of the cerebral infarction. Therefore, “therapy” in thepresent invention means treatment to reduce the effect of a blood flowdisorder after it occurs in the brain.

More specifically, a therapeutic effect of the pharmaceutical agents ormethods of this invention refers to an effect whereby the administrationof a pharmaceutical agent or the application of a method of thisinvention, after the onset of cerebrovascular disorder, reduces braintissue damage caused by the cerebrovascular disorder, compared to whennothing is administered. Therefore, “therapy” in the present inventionencompasses not only complete recovery from the damage, but also theeffect of reducing the degree of damage.

On the other hand, “prevention” in the present invention refers to areduction in the effect of a blood flow disorder, by the preventativeadministration of an HGF gene prior to the blood flow disorder occurringin the brain. More specifically, HGF gene administration is said to havea preventive effect when the administration of an HGF gene prior to theonset of a cerebrovascular disorder, such as a cerebral infarction,reduces the brain tissue damage caused by the cerebrovascular disorderthat occurs after administration, compared to when nothing isadministered. Therefore, “prevention” in the present inventionencompasses not only complete recovery from the damage, but also theeffect of reducing the degree of damage.

The terms “therapeutic agent” and “preventive agent” of this inventionare used as terms that mean pharmaceutical formulations comprising theabove-mentioned functions. The therapeutic methods and preventivemethods of this invention are methods comprising the step ofadministering a pharmaceutical formulation comprising an above-mentionedeffect.

On the other hand, cerebrovascular disorders in this invention refer toconditions in which blood flow to the brain is inhibited. Disorders thatcause inhibition of blood flow to the brain are, for example, cerebralinfarctions and intracerebral hemorrhages. Inhibited blood flow is notlimited to that caused by disease. For example, conditions of reducedblood flow resulting from a blood vessel artificially sealed off insurgical treatment, and from an injured blood vessel due to a wound, areincluded in the cerebrovascular disorders of this invention. Thecerebrovascular disorders of this invention include, for example,cerebrovascular disorders that cause ischemic or infarcted lesions inthe cerebral parenchyma, such as a cerebral infarction (cerebralthrombosis, cerebral embolism, and the like), intracerebral hemorrhage,subarachnoid hemorrhage, hypertensive encephalopathy, cerebrovasculardementia, and Alzheimer-type dementia.

The therapeutic or preventive agents of the present invention comprise asufficient amount of HGF gene to accomplish the objective intended bythe pharmaceutical agent. In other words, the agents of the presentinvention comprise an HGF gene in a “therapeutically effective amount”or a “pharmacologically effective amount”. The terms “therapeuticallyeffective amount” and “pharmacologically effective amount” are effectiveamounts of pharmaceutical agent to produce the intended pharmacologicalresults, and sufficient amounts to relieve the symptoms of the patientto be treated. Assays useful in confirming an effective dose for aparticular application are, for example, methods for measuring thedegree of recovery from a target disease. The amount that shouldactually be administered varies depending on the age, weight, andsymptom of the patient being treated, as well as on the administrationmethod and such, and is preferably an amount optimized to achieve adesired effect without marked side effects.

Therapeutically effective amounts, pharmacologically effective amounts,and toxicity can be determined by cell culture assays or optionally, byusing appropriate animal models. Such animal models can be used todetermine the desired concentration range and administration route for apharmaceutical agent. Based on these animal models, one skilled in theart can determine the effective dose for a human. The dose ratio oftherapeutic effect to toxic effect is called the “therapeutic index”,and this can be expressed as the ratio: ED50/LD50. Pharmaceuticalcompositions with a large therapeutic index are preferred. Anappropriate dose is selected according to the dosage form, the patient'ssensitivity, age, and other conditions, and the type and severity of thedisease. Although the dose of a therapeutic agent of the presentinvention differs depending on the condition of the patient, the adultdose of an HGF gene is in the range of approximately 1 μg toapproximately 50 mg, preferably in the range of approximately 10 μg toapproximately 5 mg, and more preferably in the range of approximately 50μg to approximately 5 mg. In particular, since an HGF gene can berepeatedly administered by an HVJ envelope method, administration of theHGF gene can be performed not only once, but many times, such as two orthree times, to obtain better therapeutic or preventive effects. Suchmultiple administrations using an HVJ envelope are also included in thetherapeutic or preventive methods of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of photographs of the TTC-stained coronal slices of rats(three out of six animals) belonging to the physiological saline group.

FIG. 2 is a set of photographs of the TTC-stained coronal slices of rats(three out of six animals) belonging to the pVAXI group.

FIG. 3 is a set of photographs of the TTC-stained coronal sections ofrats (three out of six animals) belonging to the HGF group.

FIG. 4 is a graph comparing the areas of the infarcted areas in thephysiological saline group, the pVAXI group, and the HGF group. Aschematic diagram of the rat brain is shown above right. The coronalsections were prepared by slicing the brains along the lines indicatedas 1 to 5. The numbers 1 to 5 in the schematic diagram correspond tothose on the horizontal axis. The vertical axis indicates the percentage(%) of the total coronal slice area that is occupied by the infarctedarea.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be specifically illustrated withreference to Examples, but is not to be construed as being limitedthereto.

(1) Production of HGF Gene Expression Vector

Human HGF cDNA (2.2 kb) was inserted between the BamHI and NotI sites ofthe pVAX1 vector (Invitrogen).

(2) Preparation of the HVJ-Envelope

Immediately prior to its use, the purified Sendai virus (HVJ) (Z strain)was inactivated by UV irradiation (99 mJ/cm²). Next, the inactivated HVJ(15,000 hemagglutination units (HAU)) and the vector produced in (1), oran expression vector not comprising HGF cDNA, were mixed with 0.3%Triton X-100 solution at 4° C. This mixture was centrifuged for 15minutes at 4° C. After removing the supernatant, buffering salt solutionwas added to the residue and then centrifuged for another 15 minutes at4° C. After removing the supernatant, the pellet was suspended in 100 μLof phosphate-buffered saline, and used to investigate the effect oncerebral infarction.

-   (3) Administration of HVJ envelope-DNA Complex to a Rat Right Middle    Cerebral Artery Occlusion Model

Wistar rats weighing 250 to 270 g were anesthetized with halothane (4%at the time of introduction; maintained at 1%). Animals were thencisternally administered, using a 26 G needle, with: 1) physiologicalsaline (100 μL) for the physiological saline group; 2) pVAXI (400μg)/HVJ-E (15,000 HAU) (100 μL) for the pVAXI group, comprising vectorsonly; and 3) pVAXI-HGF (400 μg)/HVJ-E (15,000 HAU) (100 μL) for theHGF-administered group. Six animals were used in each group. Three daysafter administration, 21 mm of 4-0 nylon suture coated withpoly-L-lysine was inserted from the right external carotid artery intothe right internal carotid artery under halothane anesthesia, to producea right middle cerebral artery occlusion model. During this treatment,the body temperatures of the animals were maintained at around 37° C.using a heating pad, and their blood pressures were measured at thecaudal artery. Neurological evaluation was carried out one hour and 24hours after this treatment. Neurological evaluation was performedaccording to the standard shown below, and the total scores of eachgroup were compared (denoted in Table 1 as the Neurological SeverityScore (NSS)). The results were statistically analyzed using aMann-Whitney U test with StadtView 5.0J.

1) Presence of Flexion of the Left Foreleg

-   -   0 Not flexed    -   0.5 Slightly flexed    -   1 Completely flexed        2) Resistance Against Lateral Compression    -   0 Same degree of resistance to the right and left    -   0.5 Slightly weakened resistance    -   1 No resistance        3) Body Posture    -   0 Normal    -   0.5 Slightly bent to the left    -   1 Completely bent to the left        4) Position of the Left Foreleg    -   0 Quickly returns to original position    -   0.5 Returns to original position    -   1 Does not return to original position        5) Position of the Right Foreleg    -   0 Quickly returns to original position    -   0.5 Returns to original position

1 Does not return to original position TABLE 1 Physio- logical HGF/HVJpVAXI/HVJ saline p-Value Body weight 250.4 ± 2.0   253 ± 2.3 251.8 ±2.4  Blood pressure 88.6 ± 1.9 88.8 ± 3.7 88.5 ± 2.1 (before treatment)Blood pressure 82.4 ± 1.7 81.2 ± 3.8 82.8 ± 1.4 (after treatment) Bodytemperature 37.4 ± 0.1 37.3 ± 0.1 37.2 ± 0.2 (before treatment) Bodytemperature 37.6 ± 0.1 37.1 ± 0.2 37.4 ± 0.2 (after treatment) NSS (1hr)  1.0 ± 0.2  1.3 ± 0.4  1.4 ± 0.2 p < 0.05* NSS (24 hr)  1.0 ± 0.1 1.4 ± 0.2  1.3 ± 0.3 p < 0.05**Compared to the physiological saline group using a Mann-Whitney U test.

As shown in Table 1, differences in blood pressure and body temperatureduring the operation were not observed between the physiological salinegroup, pVAXI group, and HGF group. The score for neurological evaluationwas significantly lower for the HGF group than the other two groups,suggesting that administration of the HGF gene maintains neurologicalfunction.

The animals were sacrificed 24 hours later, and coronal sections wereprepared every 2 mm from the frontal pole (at the positions indicated bylines 1 to 5 in the schematic diagram to the top right of FIG. 4). TTCstaining was performed, and the areas of the infarcted areas werecompared (FIGS. 1 to 3). To compare these areas, the numbers of pixelsoccupied by the infarcted areas were measured using Adobe Photoshop 5.0.The effect of the edema was taken into consideration, and evaluationswere made according to the following formula: (the lateral area of ahealthy brain−(the lateral area of an infarcted brain−area of infarctedarea))/the lateral area of a healthy brain×100 (%). FIG. 4 shows theresults. The proportion of the infarcted area compared to the total areaof the coronal section is indicated as a percentage. As is apparent fromFIGS. 1 to 4, the infarcted area in the HGF gene transfected group wasconfirmed to be reduced.

INDUSTRIAL APPLICABILITY

The above-mentioned results confirmed that the administration of an HGFgene shows advantageous effects, maintains neuronal function, andreduces the size of an infarcted area in the early stages ofcerebrovascular disorders, such as cerebral infarctions. Morespecifically, HGF was shown to probably play a role in regulatingcerebrovascular disorders. The present invention provides novel methodsfor treating cerebrovascular disorders, including cerebral infarctions,where the methods involve overexpressing HGF by introducing an HGF gene.The present methods utilizing HGF gene enable active treatment ofcerebrovascular disorders, including cerebral infarction, by genetransfer. The present methods also enable maintenance of neuronalfunction and inhibition of the infarcted area in patients for whomappropriate treatment methods did not exist until now.

1. An agent for treating or preventing a cerebrovascular disorder,wherein the agent comprises a nucleic acid encoding a protein effectiveas a hepatocyte growth factor.
 2. The agent of claim 1, wherein thecerebrovascular disorder is a cerebral infarction.
 3. The agent of claim1, wherein the agent is in the form of a tablet, pill, sugar-coatedtablet, capsule, liquid, gel, ointment, syrup, slurry, or suspension. 4.The agent of claim 1, wherein the agent further comprises a viralenvelope vector, an internal type liposome, an electrostatic typeliposome, an HVJ-liposome or improved HVJ-liposome, a cationic polymer,or combinations of two or more thereof.
 5. The agent of claim 1, whereinthe agent further comprises an HVJ-envelope.
 6. A method for treating orpreventing a cerebrovascular disorder, wherein the method comprisesintroduction of the agent of claim
 1. 7. The method of claim 6, whereinthe cerebrovascular disorder is a cerebral infarction.
 8. The method ofclaim 6, wherein the introduction of the nucleic acid comprisesintroducing the nucleic acid by an HVJ-envelope.
 9. (canceled) 10.(canceled)
 11. The method of claim 6, wherein the introduction of thenucleic acid comprises introducing the nucleic acid by viral envelopevectors, internal type liposomes, electrostatic type liposomes,HVJ-liposomes or improved HVJ-liposomes, receptor-mediated genetransfer, transfer of nucleic acid into a cell using a particle gun(gene gun), direct introduction of naked-nucleic acid, introductionusing a cationic polymer, or combinations of two or more thereof.